Petroleum is a limited, natural resource found in the earth in liquid, gaseous, or solid forms. However, petroleum products are developed at considerable costs, both financial and environmental. In its natural form, crude petroleum extracted from the Earth has few commercial uses. It is a mixture of hydrocarbons, e.g., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, etc. of varying length and complexity. In addition, crude petroleum contains other organic compounds (e.g., organic compounds containing nitrogen, oxygen, sulfur, etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.). Due to its high energy density and its easy transportability, most petroleum is refined into fuels, such as transportation fuels (e.g., gasoline, diesel, aviation fuel, etc.), heating oil, liquefied petroleum gas, etc.
Petrochemicals can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, or gels. Examples of specialty chemicals which can be produced from petrochemical raw materials include fatty acids, hydrocarbons (e.g., long chain hydrocarbons, branched chain hydrocarbons, saturated hydrocarbons, unsaturated hydrocarbons, etc.), fatty alcohols, fatty esters, fatty aldehydes, ketones, lubricants, etc. Specialty chemicals have many commercial uses. Fatty acids are used commercially as surfactants. Surfactants can be found in detergents and soaps. Fatty acids can also be used as additives in fuels, lubricating oils, paints, lacquers, candles, shortenings, cosmetics, and emulsifiers. In addition, fatty acids are used as accelerator activators in rubber products. Fatty acids can also be used as a feedstock to produce methyl esters, amides, amines, acid chlorides, anhydrides, ketene dimers, and peroxy acids and esters.
Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
There is a need for alternative routes to create both fuels and products currently derived from petroleum. As such, microbial systems offer the potential for the biological production of numerous types of biofuels and chemicals. Renewable fuels and chemicals can be derived from genetically engineered organisms (such as bacteria, yeast and algae). Naturally occurring biosynthetic pathways can be genetically altered to enable engineered organisms to synthesize renewable fuel and chemical products. In addition, microbes can be tailored, or metabolically engineered, to utilize various carbon sources as feedstock for the production of fuel and chemical products. For example, FAME (fatty acid methyl ester) can be produced by adding methanol to a culture of a recombinant host cell expressing a wax ester synthase, for example, a wax ester synthase derived from Marinobacter hydrocarbonoclasticus (“M. hydrocarbonoclasticus”). However, the expression of the wild type wax ester synthase from M. hydrocarbonoclasticus does not produce significant amounts of FAME, thus development of an improved wax ester synthase is highly desirable. In addition, the wild-type wax ester synthase from M. hydrocarbonoclasticus produces significant amounts of beta hydroxy (“β-OH”) esters, which are not typically found in fuel or chemicals derived from plant sources. See, e.g., Patent Cooperation Treaty Application No. PCT/US12/31682, which is incorporated by reference herein. Thus, it would be desirable to engineer an ester synthase to produce higher yields of fatty ester; and an ester synthase that produces higher yields of fatty ester without producing significant amounts of β-OH ester, when expressed in a recombinant host cell. Finally, it would be desirable to engineer other enzymes that have ester synthase activity to further improve the yield of fatty ester production.
Notwithstanding the advances in the field, there remains a need for improvements in genetically modified enzymes, recombinant host cells, methods and systems in order to achieve robust and cost-effective production of fuels and chemicals of interest by fermentation of recombinant host cells. The present disclosure addresses this need by providing a number of ester synthase enzyme variants that increase the yield of fatty esters. Some of the ester synthase enzyme variants of the disclosure also alter (i.e., increase or reduce) the β-OH ester content of the fatty ester composition produced by fermentation of recombinant host cells that express the ester synthase enzyme variants. The present disclosure further addresses this need by providing a number of thioesterase variants that have been engineered to have greater ester synthase activity, thereby further enhancing fatty ester production.